rhce*ce329c rhce allele associated with a weakened c ... · pdf filerhce*ce329c: a novel rhce...
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RHCE*Ce329C:A novel RHCE allele associated with a weakened C antigen expression and
genetically linked to RHD*DVII allele
Cédric VRIGNAUD1, Stéphanie RAMELET1, Michèle ROUSSEL1,
Marie-Christine SCHERMACK2, Céline NARBOUX3, Michel HENNION3,
Thierry PEYRARD1
1Institut National de la Transfusion Sanguine, Paris, France2Centre Hospitalier Régional Metz-Thionville, France
3Etablissement Français du Sang Nord de France
BBTS Annual Conference, Glasgow, September 13th – 15th 2017
• 55 antigens◦ Most known: D, C, E, c, e …
• 2 genes RHD & RHCE
• More than 500 allelic variants2Cédric Vrignaud - INTS
The Rh blood group system (ISBT 004)
2 proteins with93% of homology
• One of the activities of our referencecenter (CNRGS)◦ Discrepant and/or weakened reactivity of
one/several Rh antigens◦ Tools serology : monoclonal antibodies molecular biology : DNA chips, gDNA
or cDNA sequencing• Samples from donors and patients
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Laboratory investigation of Rh variants
24 blood samples referred for a weak ordiscrepant C antigen
◦ 3 patients (pregnant women) and 21 donors◦ From North Africa (N=21) or West Europe (N=3)
4Cédric Vrignaud - INTS
Positive with clones P3X255-13G8 + MS24Negative with clone DGC02
Donor serology (on automatedOlympus/Beckman PK7300 system)
Study background
With a set of monoclonal reagents
For C antigen
For e antigen
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Clones
MS-24(IgM,
millipore)
MS-273(IgM,
millipore)
DGCO2(IgM,
Diagast)
P3x255-13G8(IgM,
Diagast)
MS-23(IgG,
Millipore)
Reactivity 2 to 4+ 0 to 4+ 0+ 0+ 3+
Clones(IgM fromMillipore)
MS-16 MS-21 MS-62 MS-63
Reactivity 3 to 4+ 2 to 3+ 4+ 4+
Molecular study of the RHCE gene
The 1st of these cases was investigated in 2007(pregnant woman), but only exon 4 and exons 7 to9 sequencing was available at that time
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=> cDNA sequencing=> discovery of a novel
mutation: c.329T>C in RHCE
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Molecular study of the RHCE gene
All others cases wereexplored and characterizedby gDNA sequencing
One case was homozygousfor the Ce(329C) allele
No anti-C alloimmunisationhas been observed
The RH Haplotype
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Chromosome 1 (1p36)
RHD/RHCE genes on a same chromosome transmittedtogether to the offspring
RH « genetic association »
When a RH allele very often segregates with a given RH allele (in cis position).
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RHCE*Ce-D(4)-Ce allele RHD*D allele
systematicallyassociated with
Example of the RN haplotype (from Sec- subject)
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RHD allele(s) RHCE allele(s)RHD*DAU0 RHCE*ceMO
(Westhoff et al, 2013)
RHD*DAU0 orRHW1 type 4.0
RHCE*ceCF(Martin-Blanc et al, 2013)
RHD*DARRHCE*ceAR or RHCE*ceEK
(Hemker et al, 1999 ; Noizat-Pirenne et al, 2002)
RHD*DIVa-2 RHCE*ceTI(Westhoff et al, 2013)
RHD*DOL1 or DOL2RHCE*ceBI or RHCE*ceSM
(Roussel et al, 2013)
RHD*DIIIa orRHD*DIIIa-CEVS(4-7)-D or
RHD*D-CEVS(4-7)-DRHCE*ce(733G,1006T)
(Faas et al, 1997 ; Pham et al, 2009)
For every new RH allelic variant discovered ->Search for a potential association in cis
Our 24 samples were all found to show aRHD*DVII allele
RHD*DVII allele partial D (lack of epD8) c.329T>C mutation (Rouillac et al, 1995)
more frequent than DVI in France (Hennionet al, Transfus Clin Biol 2013 (Suppl))
Usually undetected by routine anti-Dtyping reagents for patients/donors
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Our 24 samples were all found to show aRHD*DVII allele
9 at heterozygous state 15 at homozygous or most probably
hemizygous state. Partial D confirmed byserological data
The homozygous Ce(329C) sample wastested for the Rhesus box (Innotrain) andconfirmed to be homozygous for theRHD*DVII allele
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22 DVII samples were available in our bloodlibrary 16 referred for a discrepant D reactivity 6 referred for anti-D immunisation in a D+subject
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RHCE*Ce329C allele RHD*DVII allele
systematicallyassociated with?
All showed a conventional RHCE*Ce allele(no c.329T>C mutation in exon 2)
CONCLUSIONS
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NovelRHCE*Ce329C allele RHD*DVII allele
geneticallyassociated with
Ancestral gene conversion event?
Another example of RHCE=>RHD genetic linkage
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RHD*DVII : detectable by only very few antibodies
RHCE*Ce329C : easily detectable by anti-C Ab
ACKNOWLEDGEMENTS
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INTS-CNRGS TeamMichèle RousselStéphanie RameletAurélien BeigengerMickael AnselmiStéphanie Martin-BlancSabrina NakesDr Thierry Peyrard
Michel HennionDr Céline NarbouxDr SchumacherDr Dumont
Dr SchermackDr Braun
THANK YOU FOR YOUR ATTENTION
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